13C MRS and LC–MS Flux Analysis of Tumor Intermediary Metabolism

Abstract

We present the first validated metabolic network model for analysis of flux through key pathways of tumor intermediary metabolism, including glycolysis, the oxidative and non-oxidative arms of the pentose pyrophosphate shunt, the TCA cycle as well as its anaplerotic pathways, pyruvate–malate shuttling, glutaminolysis, and fatty acid biosynthesis and oxidation. The model that is called Bonded Cumomer Analysis for application to 13C magnetic resonance spectroscopy (13C MRS) data and Fragmented Cumomer Analysis for mass spectrometric data is a refined and efficient form of isotopomer analysis that can readily be expanded to incorporate glycogen, phospholipid, and other pathways thereby encompassing all the key pathways of tumor intermediary metabolism. Validation was achieved by demonstrating agreement of experimental measurements of the metabolic rates of oxygen consumption, glucose consumption, lactate production, and glutamate pool size with independent measurements of these parameters in cultured human DB-1 melanoma cells. These cumomer models have been applied to studies of DB-1 melanoma and DLCL2 human diffuse large B-cell lymphoma cells in culture and as xenografts in nude mice at 9.4 T. The latter studies demonstrate the potential translation of these methods to in situ studies of human tumor metabolism by MRS with stable 13C isotopically labeled substrates on instruments operating at high magnetic fields (≥7 T). The melanoma studies indicate that this tumor line obtains 51% of its ATP by mitochondrial metabolism and 49% by glycolytic metabolism under both euglycemic (5 mM glucose) and hyperglycemic conditions (26 mM glucose). While a high level of glutamine uptake is detected corresponding to ~50% of TCA cycle flux under hyperglycemic conditions, and ~100% of TCA cycle flux under euglycemic conditions, glutaminolysis flux and its contributions to ATP synthesis were very small. Studies of human lymphoma cells demonstrated that inhibition of mammalian target of rapamycin (mTOR) signaling produced changes in flux through the glycolytic, pentose shunt, and TCA cycle pathways that were evident within 8 h of treatment and increased at 24 and 48 h. Lactate was demonstrated to be a suitable biomarker of mTOR inhibition that could readily be monitored by 1H MRS and perhaps also by FDG-PET and hyperpolarized 13C MRS methods.

Mechanism of antineoplastic activity of lonidamine

“…To quantitatively model the metabolic pathways related to energy and other intermediary metabolism, Shestov et al (32, 58) have used 13 C metabolic modeling techniques called Bonded Cumomer and Fragmented Cumomer Analysis for analysis of 13 C NMR and LC-MS data, respectively. These techniques provide a detailed picture of metabolism at the level of in vivo enzyme activities, whole pathways and the integrated systems level.…”

“…Transport of the main nutrients utilized by cancer cells were included in the model: the perfused labeled glucose, lactate and glutamine were transported from the extracellular medium to the cancer cells and vice versa assuming reversible non-steady-state Michaelis-Menten transport kinetics through corresponding transporters. Isotopomer balance equations were derived for all bonded cumomers of orders 1, 2, and 3 of participating metabolites (32, 57, 58). Fine structure multiplets were completely described by each metabolite’s bonded cumomers (32, 57, 58) of order 1, 2, and 3 using connection matrices between bonded cumomers and 13 C fine multiplets (32, 57, 58).…”

“…Isotopomer balance equations were derived for all bonded cumomers of orders 1, 2, and 3 of participating metabolites (32, 57, 58). Fine structure multiplets were completely described by each metabolite’s bonded cumomers (32, 57, 58) of order 1, 2, and 3 using connection matrices between bonded cumomers and 13 C fine multiplets (32, 57, 58). The use of bonded and fragmented cumomer techniques leads to a reduced number of equations compared to a model including all possible isotopomers while retaining all the NMR- and MS-measurable isotopomer information.…”

Metabolic Detection of Bruton's Tyrosine Kinase Inhibition in Mantle Cell Lymphoma Cells

“…We have performed a comprehensive metabolic analysis of IBRsensitive and IBR-poorly responsive MCL cells following administration of IBR: RNA-Seq analysis focusing on metabolic genes, LC/MS, and magnetic resonance spectroscopy (MRS) on intracellular metabolites and extracellular flux measurements. We have utilized 13 C tracer experiments with MRS and LC/MS combined with comprehensive and compartmentalized metabolic network analysis methods (3)(4)(5)(6) to determine intracellular fluxes through various metabolic pathways and measured contributions of these fluxes to overall tumor energy metabolism. We found high glycolysis and glutaminolysis activities and high oxidative pentose phosphate pathway (PPP) to glycolysis ratios in both IBRsensitive MCL-RL and IBR-poorly responsive JeKo-1 cells under baseline conditions.…”

The importance of accurately correcting for the natural abundance of stable isotopes

“…The traditional uses of isotope labeling experiments fall under mass isotopomer distribution analysis, a term that is sometimes used interchangeably with isotopomer enrichment assays and isotopomer spectral analysis (Buescher et al, 2015; Chubukov et al, 2013; Crown et al, 2016; Hellerstein and Neese, 1999; Kelleher et al, 1994; Mehrmohamadi et al, 2014; Papageorgopoulos et al, 1999; Revelles et al, 2013; Shestov et al, 2016; Strong et al, 1985; Wittmann, 2007). These assays can measure the kinetics of condensation reactions, such as polymeric protein and fatty acid synthesis, by adding tracers labeled with stable isotopes to catabolic biochemical pathways and tracing the transfer of isotopic labels between enriched precursors and their macromolecular products.…”